Leflunomide induces apoptosis of thymocytes and T-cell hybridoma: differences in sensitivity and signaling pathways

Abstract

LEFLUNOMIDE is a novel antiphlogistic and immunomodulating agent that has been shown to be efficacious in preventing and healing autoimmune disorders and reactions leading to organ transplantation rejection. However, the mechanisms of its action are not sufficiently elucidated. It has been postulated that the immunomodulatory effect of the drug is probably a consequence of reversible inhibition of T-cell proliferation by blocking the early stage of T-cell activation and preventing the expression of interleukin 2 receptor (IL-2R). The effect is partly dependent on inhibition of certain protein tyrosine kinases (PTK). In this work we show evidence that A77 1726, an active metabolite of leflunomide, induces apoptosis of rat thymocytes and rat thymocyte hybridoma in vitro. Experiments were done on thymocytes of AO rats (9 weeks old) and BWRT8 hybridoma cells. The following chemicals were obtained from ICN (Costa Mesa, Calif, USA); RPMI 1640 medium; fetal calf serum (FCS); Z-PheAla-CH2F, an IL-1b converting enzyme (ICE)-like inhibitor; Z-Val-Ala-Asp(Ome)-CH2F, a caspase inhibitor; and dexamethasone. Anti-rat abT-cell receptor (TCR) monoclonal antibody (mAb) was purchased from Serotec (Oxford, UK). Merocyanine (MC) 540, propidium iodide (PI), phorbol myristate acetate (PMA), genistein, and herbimycin A were obtained from Sigma (Munich, Germany). Cyclosporin A was obtained from Novartis (Basel, Switzerland), whereas A77 1726 was obtained from Hoechst, AG (Wiesbaden, Germany). Apoptosis induced by A77 1726, genistein, herbimycin A, dexamethasone, or immobilized R73 mAb was measured after cultivation of the cells for 20 hours (RPMI 1 10% FCS) using morphological criteria, MC 540, and PI staining. The percentage of specific apoptosis was determined as previously described. We found that A77 1726 induced apoptosis of both thymocytes and BWRT8 hybridoma cells, as judged by staining with MC 540 (Fig 1A), morphological analysis of nuclei (chromatin condensation, pyknosis, and kariorexis) (Fig 1B), and staining with PI (Fig 1C). All the methods gave comparable results. As shown in Fig 1A and 1B, A77 1726 at concentrations between 10 mmol/L and 100 mmol/L induced apoptosis of thymocytes in a dose-dependent manner. Approximately 10 times lower concentrations of the metabolite were necessary for apoptosis induction in BWRT8 cells. This is the first report showing direct proapoptotic effect of leflunomide on thymocytes and T-cell hybridoma. Peripheral T cells are more resistant, but the compound significantly modulates activation-induced death of these cells (Colic et al, (manuscript in preparation). It is well known that glucocorticoids and TCR crosslinking promote apoptosis of thymocytes. A similar phenomenon was published for BWRT8 cells. Therefore, in the next experiments we studied the interference of A77 1726 with dexamethasone and an mAb to rat abTCR (R73) immobilized to plastic in inducing apoptosis. As shown in Fig 1, the leflunomide metabolite increased apoptosis both of thymocytes and BWRT8 cells triggered by these agents. The finding may be relevant for selection processes in the thymus because the glucocorticoid-mediated apoptosis is supposed to be a model of “death by neglect,” whereas TCR cross-linking mimics the processes of negative selection. When we studied the effect of PMA, a PKC stimulator, and CsA, an inhibitor of calcineurin, the agents known to modulate apoptosis of thymocytes, it was found that PMA potentiated apoptosis of thymocytes but significantly inhibited apoptosis of BWRT8 cells. In contrast, CsA slightly inhibited the hybridoma cell death, but did not modulate apoptosis of thymocytes, either alone or in combination with A77 1726 (Fig 1C). PMA has been shown to either promote or inhibit apoptosis of T cells. The effect probably depends on whether this compound activates and/or subsequently depletes PKC and also on the type of cells or their activation state. In our experiments, a shortterm preincubation of the cells with PMA probably reflects activation of PKC, and the differences between the two cell types might be related to the differences in their activation state and intracellular signaling pathways. This is in agreement with the requirements for calcineurin, as shown using CsA. CsA has been found to prevent the activation-induced cell death (AICD) by inhibiting the Fas-L expression. However, it is not efficacious in modulating apoptosis of